Development of sandwich enzyme-linked immunosorbent assay for the detection of Cronobacter muytjensii (formerly called Enterobacter sakazakii)

Abstract

This study aimed to produce a polyclonal antibody against Cronobacter muytjensii (C. muytjensii, formerly called Enterobacter sakazakii) and to develop an immunoassay for its detection. The optimum production of rabbit anti-C. muytjensii immunoglobulin G (IgG) and chicken anti-C. muytjensii IgY was reached in weeks 8 and 9, respectively. Purification of rabbit anti-C. muytjensii IgG from immunized rabbit sera was accomplished using the caprylic acid and ammonium sulfate precipitation method. As a result, sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced two bands around 25 and 50 kDa, corresponding to a light and a heavy chain, respectively. The optimized conditions for sandwich enzyme-linked immunosorbent assay were using rabbit anti-C. muytjensii IgG (1 mu g/mL) as a detection antibody and chicken anti-C. muytjensii IgY (10 mu g/mL) as a capture antibody. In this assay, no cross-reactivity was observed with the other genera of pathogenic bacteria tested, which included Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes. The developed assay did not show cross-reactivity with other tested species of Cronobacter and Enterobacter genera such as C. turicensis, C. sakazakii, E. aerogenes, E. pulveris and E. helveticus. The detection limit of sandwich ELISA for C. muytjensii was found to be 2.0 x 104 colony forming units (CFU)/mL. In addition, detection of C. muytjensii in infant formula powder showed a low matrix effect on the detection curve of sandwich ELISA for C. muytjensii, the detection limit being found to be 6.3 x 104 CFU/mL. These findings demonstrate that the developed method is able to detect all strains of C. muytjensii. Hence, this ELISA technique has potent application for the rapid and accurate detection of C. muytjensii in dietary foods.